Kang Duk-Un, Lee Yong-Suk, Lee Jin-Woo
a Department of Applied Biology of Graduate School , Dong-A University , Busan , Korea.
b Department of Biotechnology , Dong-A University , Busan , Korea.
Prep Biochem Biotechnol. 2019;49(2):167-175. doi: 10.1080/10826068.2019.1566140. Epub 2019 Jan 28.
To enhance recovery yield of carboxymethylcellulase (CMCase), E. coli BL21/A-53 producing the histidine-tagged CMCase was constructed in this study. The recovery yield of the histidine-tagged CMCase using the His-tag affinity chromatography was 39.8%. The predicted molecular weight of the histidine-tagged CMCase was determined as 56,260 Da. Its K and V were 9.3 g l and 76.3 g l·min, respectively. The histidine-tagged CMCase hydrolyzed avicel, carboxymethylcellulose (CMC), filter paper, pullulan, xylan, but there was no detectable activity on cellobiose, p-Nitrophenyl-β-D-glucopyranoside (pNPG). The optimal temperature and pH for the enzymatic reaction of the histidine-tagged CMCase was 50 °C and 5.0. The histidine-tagged CMCase was enhanced by CoCl until the concentration of 100 mM, but inhibited by EDTA, HgCl, MnCl, NiCl, and RbCl. The characteristics of the histidine-tagged CMCase produced by E. coli BL21/A-53 were compared with those of CMCase without the histidine-tag of Bacillus subtilis subsp. subtilis A-53. The little changed characteristics of the histidine-tagged CMCase compared to the CMCase without a His-tag seemed to be the conformational change in the structure due to a His-tag.
为提高羧甲基纤维素酶（CMCase）的回收产量，本研究构建了产组氨酸标签化CMCase的大肠杆菌BL21/A-53。使用组氨酸标签亲和色谱法对组氨酸标签化CMCase的回收产量为39.8%。组氨酸标签化CMCase的预测分子量测定为56260 Da。其Km和Vmax分别为9.3 g/l和76.3 g/(l·min)。组氨酸标签化CMCase能水解微晶纤维素、羧甲基纤维素（CMC）、滤纸、支链淀粉、木聚糖，但对纤维二糖、对硝基苯基-β-D-吡喃葡萄糖苷（pNPG）无检测到的活性。组氨酸标签化CMCase酶促反应的最适温度和pH分别为50℃和5.0。组氨酸标签化CMCase在CoCl2浓度达到100 mM时活性增强，但受到EDTA、HgCl2、MnCl2、NiCl2和RbCl的抑制。将大肠杆菌BL21/A-53产生的组氨酸标签化CMCase的特性与枯草芽孢杆菌A-53无组氨酸标签的CMCase的特性进行了比较。与无组氨酸标签的CMCase相比，组氨酸标签化CMCase特性变化不大，这似乎是由于组氨酸标签导致结构发生了构象变化。
